Fluoride induces immune-inflammatory disorder in the kidneys via histone lysine crotonylation in vivo.

4. Discussion

The kidneys serve as the primary route for eliminating fluoride from the body. Toxicological and epidemiological reports have demonstrated that fluoride can harm kidney function. Immunologists have discovered that the kidneys, traditionally considered a non-immune organ, also contain immune cells, including lymphocytes and macrophages, which participate in the immune response. Additionally, an immuno-inflammatory imbalance plays a role in the occurrence and development of kidney injury and diseases. Therefore, in this study, we investigated the effect of fluoride exposure on the renal immune-inflammatory response and the possible underlying mechanism.

Intake of excess fluoride can impair renal structure and function. Ten weeks of 100?mg/L NaF with drinking water induced the pathologic changes including disordered arrangement of renal tubule cells accompanied by multiple tubular cell ruptures, nucleus and cell debris shedding, slightly dilated (Hu et al., 2023). In addition, our results were corroborated by the similar findings of the kidney. Kidney damage or nephrotoxicity leads to the impairment of detoxification and excretion functions, which can be confirmed by renal markers such as Cre, BUN, TP and ALB. (Malin et al., 2019). Cre is a muscle metabolite, while BUN is the main end-product of protein metabolism. Both are excreted via glomerular filtration and are commonly used to gauge kidney function (Myers et al., 2006, Wang et al., 2020). In a study by Li et al., rats treated with 100 mg/L NaF showed significant increases in serum BUN and Cre levels, reflecting renal biochemical changes and insufficiency due to NaF exposure (Li et al., 2021, Li et al., 2021). Similarly, Qujeq et al. discovered that oral administration of NaF at doses of 10, 20 and 30 mg/kg daily for 90 days resulted in a notable decrease in the average serum TP level and increased transaminase activity in adult rats, as compared to the control group (Qujeq et al., 2002). ALB, an early biomarker of kidney injury serves as a valuable tool for assessing early kidney toxicity from fluoride exposure. Zhao et al. observed that Wistar rats exposed to 50 mg/L NaF for six months exhibited reduced TP and ALB levels compared to the control group (Zhao et al., 2014). The current results demonstrated that fluoride exposure increased the serum levels of TP and ALB. The inconsistency in the current findings relative to previous reports may be related to the type of fluoridation, dose and duration. The exact cause requires further research.

One of the main mechanisms underlying fluoride toxicity is oxidative stress (Wang et al., 2022a, Wang et al., 2022b, Wang et al., 2022c, Wang et al., 2022d). Oxidative injury is defined as an imbalance between ROS production and antioxidant capacity. Excessive production of ROS is thought to activate the NF-?B pathway and result in the upregulation of pro-inflammatory mediators (Farooq et al., 2019), where ROS generate genotoxic end-products such as MDA and suppress antioxidant enzymes such as SOD and GSH (Chen et al., 2019, Dahdouh et al., 2023). MDA, a byproduct of lipid peroxidation, serves as a marker of oxidative stress and cellular damage (Karadogan et al., 2022). SOD is an important antioxidant enzyme that scavenges superoxide radicals, while GSH serves as a key cellular antioxidant and cofactor for detoxification enzymes (Ma et al., 2020). LPO reflects the extent of lipid peroxidation, which can lead to cellular dysfunction and organ failure (Ramasubramanian et al., 2021). Chen et al. (2015) found that carp juveniles exposed to NaF exhibited dose- and time-dependent decreases in SOD and GSH levels in the kidneys while the MDA level showed a dose- and time-dependent increase. Similarly, Nabavi et al. (2012) observed that male Wistar rats exposed to 600?ppm NaF experienced a significant decrease in the GSH level and catalase activity in the kidneys, along with suppressed SOD activity. Additionally, a notable elevation in LPO in the kidney tissues was observed upon NaF intoxication in rats. Consistent with these findings, the current results revealed obvious alterations in oxidative stress due to NaF treatment, including increased MDA contents and LPO levels and decreased SOD activities, GSH levels. In addition, ROS have also been involved in T cell subset differentiation by regulating IFN-? and IL-17 cytokines (Aksoylar et al., 2020). Nrf2 is a central regulator of cellular resistance to oxidative stress, which could be released and translocated to the nucleus under oxidative stress, then regulates a series of antioxidant enzymes, such as HO-1 and NQO1, and plays a pivotal role in the regulation of oxidative stress, apoptosis and cancer, among others (Che et al., 2023, Weiss-Sadan et al., 2023). Consistent with the current results, Hu et al. (2021) found that 100?mg/L NaF changed the gene and protein expression levels of Nrf2, HO-1 and NQO1. These findings suggest that fluoride can induce oxidative injury.

The kidneys contain dendritic cells, macrophages, T cells and B cells. These cells participate in the immune response and play a critical role in the pathogenesis of kidney disease (Qu and Jiao, 2023). CD4⁺T cells are crucial for immune functions. They maintain host health and prevent the occurrence and development of diseases (Sun et al., 2023). CD4⁺T cells can also differentiate into Th1 or Th2 cell subsets. T-bet is known to promote the differentiation of Th1 cells and the production of pro-inflammatory cytokines such as IFN-?, which can exacerbate inflammation and tissue injury (Shang et al., 2024). GATA3 is crucial for the differentiation of Th2 cells and the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, which play key roles in tissue repair and the resolution of inflammation (Li et al., 2021, Li et al., 2021). NaF induced a significant decrease in the percentages of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ T lymphocytes and CD4⁺/CD8⁺ ratio in the peripheral blood and spleen (Guo et al., 2017; Li et al., 2021, Li et al., 2021). Peres et al. (2023) also found that CD4⁺ T cells were activated in subclinical acute kidney injury followed by Th1 and Th2 phenotype differentiation, characterized by an increase in the production of IFN-? and no change in the IL-4-producing Th2 phenotype in the renal cortex. Another study by Zhang et al. (2023) investigated the absolute T lymphocyte count and Th1/Treg cell imbalance in the peripheral blood of patients with end-stage renal disease. In the current study, the mRNA levels of the Th1 transcription factor T-bet and cytokine IFN-? were dramatically increased while the Th2 transcription factor GATA3 and cytokine IL-4 levels were significantly downregulated in the kidneys after fluoride exposure. Taken together, it appears that fluoride increases the percentages of CD4⁺T cell and induces abnormalities in Th1/Th2 cells, including polarization to Th1 subpopulations. These findings provide a deeper understanding of the fluoride-induced renal immune imbalance.

Macrophages are one of the most effective immune defence cells due to their strong ability to phagocytize and kill pathogens. They are polarized into two distinct functional phenotypes: proinflammatory (M1) and anti-inflammatory (M2) (Funes et al., 2018). CD11b is indicative of the macrophage differentiation status. CD80, expressed by macrophages and other antigen-presenting cells, serves as a marker of M1 polarization, whereas CD206 is the predominant marker for M2 macrophages, which are involved in tissue repair and anti-inflammatory processes (Shapouri-Moghaddam et al., 2018). Ma et al. (2016) found that 50??M NaF significantly enhanced CD80 expression in splenic B cells by flow cytometric analysis. Du et al. (2022) found that 5 and 10?mM NaF exposure for four weeks resulted in an increase in the M1 macrophage marker CD86 and downregulation of the number of CD206-positive cells by immunohistochemical staining. Similarly, in the current study, there were obvious increases in CD11b and CD80 expression, as well as a decrease in CD206 in the kidneys. These results suggest that fluoride suppresses M2 differentiation but augments M1 differentiation of macrophages in the kidneys.

Chronic inflammation plays a crucial role in many renal diseases, including glomerulonephritis (Levey and Coresh, 2012), tubulointerstitial nephritis (Joyce et al., 2017) and diabetic nephropathy (Foresto-Neto et al., 2024). The NLRP3 inflammasome is a multiprotein complex (NLRP3, caspase-1 and ASC) that promotes the maturation and secretion of pro-inflammatory cytokines such as IL-1? (Fu and Wu, 2023). It triggers inflammasome assembly and caspase-1 activation in response to cellular stress and damage, playing an important role in initiating and perpetuating inflammatory responses (Xu and Núñez, 2023). Li et al., 2024, Li et al., 2024 found that exposure to 100?mg/L fluoride significantly increased the mRNA and protein expression of NLRP3, caspase-1 and IL-1? in the testes of mice. In a diabetic nephropathy model induced by streptozocin (STZ) in rats, Wang et al. (2012) found that the accumulation of uric acid and lipids led to the activation of the NLRP3 inflammasome followed by the maturation and secretion of IL-1? and IL-18, which triggered further pro-inflammatory events, thus participating in the renal damage caused by STZ-induced diabetes in rats (Santoyo-Sanchez et al., 2013). In the current study, upregulation of NLRP3, caspase-1 and IL-1? was observed in the kidneys after fluoride exposure. These findings highlight the involvement of NLRP3 inflammasome activation in fluoride-induced renal inflammation.

TNF-a is known to promote inflammation and tissue damage by inducing the expression of adhesion molecules and pro-inflammatory cytokines (Zelová and Hošek, 2013). TGF-a is a potent profibrotic cytokine that contributes to tissue fibrosis and extracellular matrix deposition (Peng et al., 2022). IL-6 is a pleiotropic cytokine involved in immune regulation, the acute-phase response and tissue repair (Yu et al., 2022). IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family; it regulates both Th1 and Th2 responses (Vecchié et al., 2021). Owumi et al. found that adult male Wister rats exposed to 15 mg/L of NaF for 14 days had significantly elevated renal levels of NO, IL-1? and TNF-a (Owumi et al., 2019). In the study by Luo et al., ICR mice exposed to NaF at doses exceeding 12 mg/kg for 42 days showed alterations in the mRNA and protein expression levels of cyclooxygenase-2 (COX-2), TNF-a, IL-1B, IL-6 and interleukin-8 (IL-8) in the kidneys (Luo et al., 2017). In addition, increased secretions of proinflammatory factors including TNF-a, IL-6 and monocyte chemoattractant protein-1(MCP-1) also found in hyperuricemic nephropathy mice (Ren et al., 2021). Consistent with these studies, the current study found upregulation of inflammatory cytokines, including TNF-a, TGF-B and IL-6, in the kidneys following fluoride exposure. These results suggest that the activation of the NLRP3 inflammasome and excessive release of inflammatory factors could be involved in renal inflammation induced by fluoride.

Histone lysine crotonylation (Kcr) was first reported in 2011 in the top journal Cell by Zhao’s team at the University of Chicago (Tan et al., 2011). It also modified proteins including important regulators of key processes such as cell cycle progression, chromatin remodeling, organization, and metabolic activity (Zhao et al., 2023). Growing evidence indicates that Kcr participates in renal injury and disease. Recent studies support the chromodomain Y-like transcription repressor and crotonyl-CoA hydratase CDYL could regulate its catalytic activity on histone Kcr, and thereby modulates disease progression in autosomal dominant polycystic kidney disease (ADPKD) (Dang et al., 2022). Ruiz-Andres et al. (2016) found that histone Kcr levels and Sirt3 mRNA expression were markedly increased in the kidneys of an acute kidney injury (AKI) animal model. Yang et al. (2021) found that T. gondii infection leads to an increase in HDAC2, which reduces histone crotonylation, particularly on H2BK12cr, and suppresses NF-?B activation, impacting macrophage proliferation. Histone acyltransferase P300/CBP can transfer the crotonyl group to the ?-amino group of a lysine residue, thereby regulating gene transcription, and Sirt and HDAC are also modifying enzymes of histone Kcr (Xu et al., 2017). In the study by Suzuki and Bartlett (2014), rats treated with fluoride (0, 50, and 100 ppm) in drinking water for six weeks demonstrated significantly increased Sirt1 expression and Sirt1 phosphorylation, resulting in the augmentation of Sirt1 deacetylase activity in ameloblast-derived cells. HDAC3 has been reported to regulate TIMAP (TGF?-inhibited membrane-associated protein), thereby affecting macrophage M2 polarization markers, migration and phagocytosis (Yang et al., 2017). Wang et al. (2024) used HDAC3^-/- mice to confirm that HDAC3 can regulate the expression of proinflammatory cytokines (TNF-a, IL-1B, IL-6) and the number of CD45-marked bone marrow-derived leukocytes and F4/80-marked macrophages in the kidneys. Another study by Marumo et al. (2010) found that HDAC2 was upregulated in response to kidney injury and contributed to the induction of CSF-1, which, in turn, promoted macrophage infiltration and fibrotic responses. HDAC inhibition reduced CSF-1 levels, thereby decreasing macrophage infiltration and the associated inflammation and fibrosis in tubulointerstitial injury. In addition, HDAC1/HDAC2 can regulate CD4 lineage integrity and control CD4⁺ CTL differentiation (Preglej et al., 2020). In addition, Guan et al. found that the HDAC activity induce NLRP3 re-expression, which may be associated with HDAC-mediated chromatin condensation (Guan et al., 2024). These studies suggest that HDAC2 and HDAC3 are key regulators of kidney inflammation and immune responses. P300 is the first reported protein with histone crotonyltransferase activity (Xu et al., 2017). Liu et al. (2013) found that p300 affects Treg homeostasis or function, thereby promoting antitumour immunity. It has been reported that P300/CBP could regulate T cell lineages-related transcription factors T-BET, GATA3 and NFAT, as well as affect promoters of the pro-inflammatory cytokines such as IL-2, IL-4, and IFN-y, play an important role in T cell activation (Picavet et al., 2024). The present experimental results showed that fluoride decreased the level of the histone crotonyltransferase P300 protein and upregulated the levels of the histone decrotonylation Sirt1 and the HDAC2 and HDAC3 proteins, thereby decreasing H2BK12cr and H4K8cr in the kidneys, and this process may be associated with fluoride-induced renal damage. However, the underlying regulation mechanism of histone lysine crotonylation about fluoride-induced renal toxocity should be validated in further studies.